Ultrasound contrast agents (UCA) have been widely used in diagnostic applications in medical research and clinical practice. Typically, said UCA are in the form of stabilized gas-bubbles, also known as gas-filled microvesicles. Gas-filled microvesicles are in general divided into two main categories, i.e. microbubbles and microcapsules (or microballoons).
Microbubbles include aqueous suspensions in which the bubbles of gas are bounded at the gas/liquid interface by a thin envelope (film) involving a stabilizing amphiphilic material disposed at the gas to liquid interface. Microbubbles suspensions are typically prepared by contacting powdered amphiphilic materials, e.g. freeze-dried preformed liposomes or freeze-dried or spray-dried phospholipid solutions, with air or other gas and then with an aqueous carrier, while agitating to generate a microbubble suspension which can then be administered, preferably shortly after its preparation. Examples of aqueous suspension of gas microbubbles and preparation thereof are disclosed, for instance, in U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,556,610, U.S. Pat. No. 5,597,549, U.S. Pat. No. 5,827,504 and WO 04/069284.
Microcapsules (or microballoons) include suspensions in which the bubbles of gas are surrounded by a solid material envelope of a lipid or of natural or synthetic polymers. The thickness of microcapsules envelope may vary from few nanometers to few hundreds of nanometers. Examples of microcapsules and of the preparation thereof are disclosed, for instance, in U.S. Pat. No. 5,711,933 and U.S. Pat. No. 6,333,021.
Recently, these UCA have been exploited in ultrasound-mediated gene delivery. This novel approach uses acoustic cavitation effects of gas microvesicles to induce transient membrane permeabilization at the cellular level (also known as “sonoporation”), thereby facilitating the transfer of drugs or genetic materials into the cell. Gas-filled microvesicles compositions have thus been developed, where the microvesicles contain, either in their internal void portion or embedded into the stabilizing envelope, a therapeutic compound. For instance, U.S. Pat. No. 6,416,740 discloses a targeted therapeutic system comprising gas-filled microspheres incorporating a therapeutic compound. Similarly, US Patent application no. 2004/0258760 relates to polymeric microcapsules loaded with a bioactive compound. This solution has however the disadvantage that the therapeutic compound has to be somehow inserted into the or attached to the gas-filled microvesicle, thus entailing cumbersome manufacturing processes. In addition, the fact that same microvesicle carrying the therapeutic compounds shall undergo to the cavitation effects to provide the necessary cellular permeabilization renders the whole system rather ineffective. Furthermore, as the therapeutic compound is in general hydrophobic or has to be rendered hydrophobic (e.g. in the case of genetic material) for it to be compatible with the material forming the microvesicles, the release of the therapeutic compound may be less effective in the hydrophilic environment of the blood circulation or of the cells.
The Applicant has now found that the use of a composition comprising gas-filled microcapsules having a predetermined resistance to the mechanical index of the applied ultrasound wave notably enhances the effectiveness of the delivery of therapeutic compounds (such as genetic material) into the cell, particularly when said therapeutic compound material is substantially free of any stable binding with the shell of said microcapsules.